PROJECT SUMMARY/ABSTRACT Mediator is a conserved, essential regulator of RNA Polymerase II (RNAPII) transcription that is proposed to function as a connector between distal regulatory elements and gene-proximal promoters. This bridging depends on two distinct modules of Mediator: the tail, which interacts with transcriptional activators bound to specific DNA sequences at a distal regulatory element, and the head, which interfaces with the promoter- associated basal transcription machinery. A major outcome of this bridging activity of Mediator is thought to be a positive impact on the formation of the RNAPII pre-initiation complex (PIC). Using ChEC-seq, a method that we introduced for global mapping of protein-DNA interactions, we elucidated a role for Mediator in the recruitment of the TFIID complex, a component of the PIC, to the majority of promoters in the yeast genome. We propose to expand on this finding via a thorough characterization of the role of Mediator in the recruitment of each subcomponent of the PIC, which will yield a comprehensive view of the role of Mediator in PIC formation in vivo. While composed of over two dozen subunits, Mediator has been proposed to be generally monolithic. However, there is evidence that the tail module might have functions independent of the complete Mediator complex, though this has not been rigorously tested. We will investigate the transcriptional effects of severing the Mediator tail from the complex as well as the transcriptional consequences of removing the tail after it has been severed. Lastly, we propose to expand our studies of Mediator beyond yeast and into mammalian cells, where Mediator associates with distal enhancer elements. While the association of Mediator with enhancers is well known, it is unclear if its role as a transcriptional regulator extends to the transcription of enhancer RNAs (eRNAs). We will thus determine the effects of Mediator depletion on eRNA transcription as well as PIC formation at enhancers. The critical role of Mediator in transcriptional regulation is underscored by the numerous human disorders linked to its dysregulation: point mutations in various Mediator subunits have been implicated in several neurodevelopmental disorders, and alterations in Mediator subunit expression have been observed in a wide variety of cancers. The fundamental insights into Mediator function gained through this work will therefore enhance not only our understanding of the core functions of Mediator but also why its dysregulation is so often observed in disease.